Animal and plant cells have the capacity to eliminate a diversity of lipophilic toxins from the cytosol following conjugation of the toxin with glutathione (GSH) (Ishikawa et al., 1997, Bioscience Reports. 17:189-208; Martinoia et al., 1993, Nature 364:247-249; Li et al., 1995, Plant Physiol. 107:1257-1268). This process is mediated by the glutathione S-conjugate (GS-X) pumps which are novel MgATP-dependent transporters that catalyze the efflux of GS-conjugates and glutathione disulfide (GSSG) from the cytosol via the plasma membrane and/or endomembranes. GS-X pumps are considered to constitute a terminal phase of xenobiotic detoxification in animals and plants.
The metabolism and detoxification of xenobiotics comprises three main phases (Ishikawa, 1992, supra). Phase I is a preparatory step in which toxins are oxidized, reduced or hydrolyzed to introduce or expose functional groups having an appropriate reactivity. Cytochrome P450 monooxygenases and mixed function oxidases are examples of phase I enzymes. In phase II, the activated derivative is conjugated with GSH, glucuronic acid or glucose. In the case of the GSH-dependent pathway, S-conjugates of GSH are formed by cytosolic glutathione-S-transferases (GSTs). In the final phase, phase III, of the GSH-dependent pathway, GS-conjugates are eliminated from the cytosol by the GS-X pump.
The GS-X pump is unique in its exclusive use of MgATP, rather than preformed transmembrane ion gradients, as a direct energy source for organic solute transport. Although an understanding of the constituents of GS-X pumps is relevant to an understanding of the mechanism by which cells combat, for example, chemotherapeutic agents and herbicides, there has until recently been a paucity of information on the molecular identity of GS-X pumps, particularly in plants.
A 190 kDa membrane glycoprotein encoded by the human multidrug resistance-associated protein gene (MRP1) has been implicated in the resistance of small cell lung cancer cell lines to a number of chemotherapeutic drugs (Cole et al., 1992, Science 258:1650-1654). This glycoprotein catalyzes the MgATP-dependent transport of leukotriene C.sub.4 and related glutathione-S-conjugates (Leier et al., 1994, J Biol. Chem. 269:27807-27810; Muller et al., 1994, Proc. Natl. Acad. Sci. USA 91:13033-13037; Zamam et al., 1995, Proc. Natl. Acad. Sci. USA 92:7690-7694).
MRP1 is a member of the ATP binding cassette (ABC) superfamily of transporter proteins. Distributed throughout the major taxa, ABC transporters catalyze the MgATP-dependent transport of peptides, sugars, ions and lipophiles across membranes. ABC transporters comprise one or two copies each of two basic structural elements, a hydrophobic integral membrane sector containing approximately six transmembrane a helices and a cytoplasmically oriented ATP-binding domain known as a nucleotide binding fold (NBF) (Hyde et al., 1990, Nature 346:362-365; Higgins, 1995, Cell 82:693-696). The NBFs are a diagnostic feature of ABC transporters and are 30% identical between family members over a span of about 200 amino acid residues, having two regions known as a Walker A and a Walker B box (Walker et al., 1992, EMBO J 1:945-951), and also having an ABC signature motif (Higgins, 1995, supra).
ABC family members in eukaryotes include mammalian P-glycoproteins (P-gps or MDRS), some of which are implicated in drug resistance and others in lipid translocation (Ruetz et al., 1994, Cell 77:1071-1081), the pleiotropic drug resistance protein (PDR5) and STE6 peptide mating pheromone transporter of yeast, the cystic fibrosis transmembrane conductance regulator (CFTR) Cl.sup.- channel, the malarial Plasmodium falciparum chloroquine transporter (PFMDR1) and the major histocompatibility (MHC) transporters responsible for peptide translocation and antigen presentation (Balzi et al., 1994, J Bioenerg. Biomemb. 27:71-76; Higgins, 1995, supra).
Sequence comparisons between MRP1 and other ABC transporters reveal two major subgroups among these proteins (Cole et al., 1992, supra; Szczypka et al., 1994, J Biol. Chem. 269:22853-22857). One subgroup comprises MRP1, the Saccharomyces cerevisiae cadmium factor (YCF1) gene, the Leishmania P-glycoprotein-related molecule (Lei/PgpA) and the CFTRs. The other subgroup comprises the multiple drug resistance proteins (MDRs), MHC transporters and STE6.
The invention described herein relates to bioremediation (specifically phytoremediation), plant responses to herbicides, plant-pathogen interactions and plant pigmentation.
With respect to bioremediation, the massive global expansion in industrial and mining activities during the last two decades together with changes in agricultural practices, has markedly increased contamination of groundwaters and soils with heavy metals. Indeed, it is estimated that the annual toxicity of metal emissions exceeds that of organics and radionuclides combined (Nriagu et al., 1988, Nature 333:1340138). Since soil and water contamination results in the uptake of heavy metals and toxins by crop plants, and eventually humans, there remains a need for a means of manipulating the ability of a plant to sequester compounds from the soil in order to better manage soil detoxification through bioremediation using native species or genetically engineered organisms.
Regarding herbicides, these compounds are generally low molecular weight, lipophilic compounds that readily penetrate cells in a passive manner. Having entered cells, herbicides inhibit plant-specific processes such as photosynthetic electron transport (e.g., atrazine, chlortoluron) or the biosynthesis of essential amino acids (e.g., glyphosate, chlorsulfuron or phosphotricine), porphyrins (e.g., acidofluorfen), carotenoids (e.g., norflurazon), fatty acids (e.g., diclofop) or cellulose (e.g., dichlobenil) (Boger et al., 1989, Target Sites of Herbicide Action, CRC Press, Boca Raton, Fla.; Devine et al., 1993, Physiology of Herbicide Action, Prentice Hall, Englewood Cliffs, N.J.). Plants that are naturally tolerant of certain herbicides either contain a cellular target that does not interact with the herbicide, have efficient systems for inactivation of the herbicide, or have a high capacity for excluding or eliminating the herbicide from the target.
Herbicide metabolism comprises the three phases described above for general xenobiotic metabolism. The first two phases (the first being oxidation and hydrolysis and the second being conjugation with GSH or glucose) contribute to detoxification by decreasing the intrinsic biochemical activity of the herbicide and/or by increasing its hydrophilicity. These two phases render the herbicide less mobile in the plant. The third phase (compartmentation) is often critical for sustained detoxification since the conjugates themselves may interfere with metabolism. For example, the herbicide synergist tridiphane, is converted to its corresponding GS-conjugate in plants to generate a potent inhibitor of atrazine metabolism. (Lamoureux et al., 1986, Pestic. Biochem. Physiol. 26:323-342).
Likewise, and more generally, GS-conjugates of any given herbicide would be expected to act as end-product inhibitors of GSTs and thereby impair long-term detoxification unless they are removed from the intracellular compartment, usually the cytosol, in which they are formed. Since the vacuolar GS-X pump of plants is known to transport several GS-herbicide conjugates, for example, those of the chloroacetanilide herbicides (metolachlor) and triazines (simetryn) (Martinoia et al., 1993, supra; Li et al., 1995, supra), there is a long felt need for a knowledge of thy molecular identity of this transporter or family of transporters. Such knowledge will enable the development of new strategies for increasing or decreasing the resistance of plants to herbicides.
With regard to plant-pathogen interactions, a key event in the disease resistance response of legumes is the rapid and localized accumulation of isoflavonoid phytoalexins. The majority of the research on plant-pathogen interactions has centered on the enzymology and molecular biology of the isoflavonoid biosynthetic pathway (Dixon et al., 1995, Physiol. Plant 93:385). However, the mechanism and sites of intracellular accumulation of these compounds is not understood. Since many isoflavonoid phytoalexins are as toxic to the host plant as they are to its pathogens, the discovery of the molecular mechanism by which these compounds are sequestered within a plant is crucial to the development of plants with increased pathogen resistance.
With regard to plant pigmentation, functional analyses of the maize gene, Bronze-2, which participates in anthocyanin pigment biosynthesis, suggest that one of the endogenous substrates for the plant vacuolar GS-X pump are anthocyanin-GS conjugates (Marrs et al., 1995, Nature, 375:397-400). Anthocyanins share a common biosynthetic origin and core structure based on cyanidin-3-glucoside. It is through the species-specific decoration of cyanidin-3-glucoside by hydroxylation, methylation, glucosylation and acylation that the wide spectrum of red, blue and purple colors in the vacuoles of flowers, fruits and leaves is produced. The molecular nature of the plant GS-X pump which mediates transport of anthocyanin-GS conjugates was not known in the art until the present invention. There remains a need to determine the molecular nature of the GS-X pump responsible for transport of anthocyanin-GS conjugates in order that plant coloration may be manipulated at the molecular level.
The present invention satisfies the aforementioned needs.